Drugs that block the rapid delayed rectifier K+ current (Ikr) cause prolongation of cardiac action potentials and electrical refractoriness. These compounds were developed as antiarrhythmic agents based on positive findings in canine models of ischemia-induced ventricular tachycardia and fibrillation. Unfortunately, most class III antiarrhythmic drugs, as well as 70 other common mediations that block Ikr as a side-effect, can cause an inhomogeneous prolongation of ventricular action potentials and induce long QT syndrome and its associated ventricular arrhythmia, torsades de pointes. It is unclear why so many structurally diverse compounds block Ikr, but this undesirable side effect is now recognized as a major hurdle in the development of new and safe drugs. The recent awareness that block of Ikr can induce arrhythmias prompted interest in the development of IKs blockers as antiarrhythmic agents. However, in the past 5 years it was discovered that mutations in any of the genes that encode the alpha- and beta-subunits that co assemble to form Ikr (HERG and MiRPl or IKs (KvLQT1 and minK) channels cause inherited long QT syndrome and sudden death. The overall goal of this project arises from our progress during the past four years where we defined the mechanisms of Ikr block and the molecular determinants of binding of a potent class III antiarrhythmic agent to the HERG channel. We now propose to characterize the blocking mechanisms and binding site for additional antiarrhythmic agents and other commonly used medications that block Kr and Ks channels. The specific aims are to characterize the molecular determinants of high affinity drug block of HERG and KvLQT1 channels, the role of the inactivated state in drug block of HERG channels, and how binding of accessory beta-subunits (minK, MiRP1 and MiRP2) enhances drug block of HERG and KvLQT1 channels. An understanding of the molecular determinants of drug binding to Kr and Ks channels will facilitate design of safer drugs that are devoid of the propensity to induce the long QT syndrome and potentially fatal arrhythmias.